African Journal of Agricultural Research Vol. 4 (9), pp.

898-902, September, 2009

Available online at http://www.academicjournals.org/AJAR
ISSN 1991-637X 2009 Academic Journals

Full Length Research Paper

Flavonoids from mulberry leaves by microwave-

assisted extract and anti-fatigue activity
Wei Li 1*, Tao Li2 and Keji Tang1
1
Dezhou University, Dezhou, Shandong Province, 253023, Peoples Republic of China.
2
China West Normal University, Nanchong, Sichuan Province, 637002, Peoples Republic of China.
Accepted 1 August, 2009

Flavonoids from mulberry leaves (FML) were extracted using microwave-assisted extraction method
(MAE). Several correlation factors were studied to optimise conditions at laboratory scale. The research
results indicated that the optimal extraction parameters are as follows: ethanol concentration is 60%,
material/solvent ratio is 1:15, microwave irradiation power is 560 W and irradiation time is 5 min. FML
were tested in mice weight-loaded swimming model. Both high-dose and middle-dose of FML could
obviously prolong the swimming endurance time, and had active effects on the serum blood urea
nitrogen (BUN), hepatic glycogen and blood lactic acid levels in mice. The above results suggest that
appropriate dose of FML possessed an anti-fatigue activity in mice.

Key words: Flavonoids, mulberry leaves, microwave-assisted extract, anti-fatigue.

INTRODUCTION

Morus alba L. (mulberry), a woody plant belonging to the
genus Morus, family Moraceae, is a widely distributed
plant in China, whose leaves, root bark and branches
have long been used in Chinese medicine to treat fever,
protect the liver, improve eyesight, facilitate discharge of
urine and lower blood pressure (Chen and Li, 2007; Jia et
al., 1999). The mulberry leaves are rich in flavonoids,
alkaloids and polysaccharides components which are
known as the most potently major active compounds by
chemical constituent investigations (Wang et al., 2008).
Among those, the flavonoids in mulberry leaves were
contained rutin, quercetin, isoquercitrin and quercetin 3-
(6-malonylglucoside) (Lee et al., 2007). The health
benefits of flavonoids are well known and are displayed
as a remarkable range of biochemical and pharmaco-
logical properties that may significantly affect the function
of various mammalian cells (Middleton et al., 2000). The
anti-inflammatory, antioxidant, antithrombotic and anticar-
cinogenic effects of flavonoids are some of the properties
that have been under consideration in view of therapeu-
tical purposes for several human diseases (Snijman et
al., 2007).
Flavonoids are thought to be one of the most critical
constituents in mulberry that have therapeutic activity.
*Corresponding author. E-mail: liweidezhouuv@126.com,
liweidezhouuv@sina.com. Tel: +86-0534-2969004.
But the anti-fatigue effects of flavonoids from mulberry
leaves (FML) havent been reported.
Conventional methods for the extraction of natural
products from plant material, eg Soxhlet, liquid-liquid, and
solid-liquid extractions are characterized by the con-
sumption of large volumes of solvent and energy, lengthy
extraction procedures, and the potentially deleterious
degradation of labile compounds (Kerem et al., 2005). In
recent years, new extraction techniques have been
developed to reduce the volume of solvent needed for
extraction (or to eliminate its use entirely), to reduce
extraction and extract clean-up times, and to improve the
reproducibility of compound recovery. These recent ex-
traction techniques include accelerated solvent extraction
(ASE), supercritical fluid extraction (SFE), solid-phase
microextraction (SPME), extraction with supercritical or
subcritical water, and microwave-assisted extraction
(MAE) (Barnabas et al., 1994; Buchholz and Pawliszyn,
1994; Hawthorne and Miller, 1994; Heemken et al., 1997;
Llompart et al., 1997; Tian et al., 2008; Mandal et al.,
2007). Most of these methods have similar pros and cons
with regard to solvent volume, extraction time and extrac-
tion efficiency. MAE is a process of using microwave
energy to heat solvents and specific molecules in contact
with a sample to enhance the extraction of soluble
components contained therein. Its main advantages are
reduced solvent volume and time consumption, and
increased sample throughput (Shu et al., 2003). This
method has been employed for the extraction of environ-

mental matrices and natural products such as essential
oils from plant materials (Chen and Spiro, 1995; Tu and
Bi, 2005), glycyrrhizic from licorice root (Pan et al., 2000),
taxans from Taxus biomass (Mattina et al., 1997), and
azadirachtin-related limonoids from neem (Dai et al.,
1999).
In the present study, the objectives were to develop a
microwave-based method for the extraction of flavonoids
from mulberry leaves (FML), and to determine whether
the MAE extract exhibits anti-fatigue activity.
MATERIALS AND METHODS
Plant material
Mulberry leaves were collected during the summer season in
Shandong Province, China. Identification of plant was verified by
associate professor Jianlin wang, Dezhou University. Fresh, intact,
viridity Mulberry leaves was picked to shade dried as experimental
material.
Microwave assisted extraction (MAE)
Experiments were carried out in a domestic microwave oven (Set
power: 280, 420, 560 and 700 W, Galanz Co., Guangdong, China).
The shade dried Mulberry leaves was crushed in an electrical
grinder and then powdered. Out of this powder, 100 g was mixed
with ethanol solvents in suitable ratio. The suspension was radiated
in microwave oven at regular intervals (one minute radiation and
two minutes off) to keep temperature not rise above 100C (Quan et
al., 2006). The extract was obtained which was filtrated by using the
filter and was evaporated by using a rotary evaporator (RE52AA,
Yalong Biochemical Instrument Co., Shanghai, China) under re-
duced pressure at 40C to get the flavonoids. It was stored at (0 -
4C) until used.
Determination of total flavoniods content
The contents of flavonoids were determined by the NaNO2-
Al(NO3)3-NaOH colorimetric assay and by reference to Rutin, and
wavelenth in spectrophotometer (V-5100, Beijing Chenxiyong-
chuang Science and Technology Co., Beijing, China) was set at 510
nm (Xu, 2007; Wang et al., 2004).
Selection of animals
Male mice of original Kun-ming strain (Dezhou University,
Shandong, China), weighing 20 2 g, which were housed in colony
cages (eight mice per cage) at an ambient temperature of 25 2C
with 12 h light and 12 h dark cycle. The mice were fed normal diets
purchased commercially from vendors. The normal diets consists of
corn starch 40%, bran 20%, soybean oil meal 20%, fish flour 14%,
corn oil 2.0%, mineral mix 3.5%, salt 0.4%,Vitamin mix 0.1%. The
animals were allowed to acclimatize to the laboratory environment
for 1 week and then randomly divided into four groups equally
based on body weight as given below:
i) Control group (CG) - mice administered water daily for 28 days;
ii) Low-dose group (LG) - mice administered FML (5 mg/kg) daily for
28 days;
iii) Middle-dose group (MG) - mice administered FML (10 mg/kg)
daily for 28 days;
iv) High-dose group (HG) - mice administered FML (20 mg/kg) daily
for 28 days.
Li et al. 899
All the experiments with animals were carried out according to
the guidelines of the institutional animal ethical committee and had
prior approval.
Anti-fatigue activity
The anti-fatigue activity of FML was evaluated by the weight-loaded
swimming model in mice. The model was a reliable measure of anti-
fatigue treatment as established in both laboratory animals and
humans (Orlans, 1987; Lapvetelainen et al., 1997). Changes in the
body weight of mice during initial, intermediate and terminal stages
of the test, Swimming endurance time and corresponding
biochemical parameters including blood lactate, serum blood urea
nitrogen(BUN) and hepatic glycogen were observed (Technical
Standards for Testing and Assessment of Health Food, Ministry of
Health, PR China, 2003).
Swimming test was applied to the mice at the end of the 28 days.
Mice were fasted for 12 h (over night) and FML was administered
orally .The size of swimming pool was designed as 50 cm (length)
50 cm (width) 40 cm (depth), filled with fresh water at 30 1C.
Eight male mice were taken out from each group to make
swimming endurance exercise. A lead block (5% of body weight)
was loaded on the tail root of the mouse. The end point of the
swimming endurance was taken as when the mouse remained at
the bottom for more than 10 s (Shi et al., 2004; Xie et al., 2004; Li
and Wen, 2006; Zhang et al., 2008; Qian et al., 2008). Swimming
endurance time for each animal was recorded.
Eight male mice were taken out from each group for blood lactate
analyses. A lead block (2% of body weight) was loaded on the tail
root of the mouse. 20 L of blood were collected from the veins of
the tails of mice after the last administration of FML. Another 20 L
of blood samples were collected immediately after mice had been
swimming for 30 min.
The levels of blood lactate were determined using a commercial
diagnostic kit obtained from Jiancheng Diagnostic Systems
(Nanjing, China).
Eight male mice were taken out from each group for hepatic
glycogen and serum blood urea nitrogen (BUN) analyses. The mice
made swimming exercise for 90 min without a load. After an hours
resting, the mice were killed to collect liver and plasma samples.
The levels of hepatic glycogen and serum BUN were determined
using commercial diagnostic kit obtained from Jiancheng Diagnostic
Systems (Nanjing, China).
Statistical analysis
Data were assessed using SPSS program (version 15.0, SPSS
Inc., Chicago, IL, USA). P values of < 0.05 between mean values
were considered statistically significant.
RESULTS AND DISCUSSION
Effect of ethanol concentration on extraction yield of
FML
Ethanol was employed in experiment because it is non-
toxic and inexpensive solvent. Ethanol concentration is
an important factor affecting extraction yield of flavonoids
(Lan et al., 2007; Su et al., 2007), which was set at 20,
40, 60, 80 and 100%, respectively. Figure 1 shows the
result of flavonoids extracted from leaves. When ethanol
concentration increased from 20 - 60%, extraction yield
900 Afr. J. Agric. Res.

2.5 2.5
Extraction yield (%)

2 2

Extraction yield (%)

1.5 1.5

1 1

0.5 0.5

0 0
20 40 60 80 1 00 280 420 560 7 00
Ethanol concentration (%) Microwave irradiation power (W)

Figure 1. E ect of ethanol concentration on extraction Figure 3. Effect of Microwave irradiation power on
yield of FML. extraction yield of FML.

2.5 1:15 - 1:25. This is due to the fact that the increasing
material/solvents ratio could decrease solution concen-
2
Extraction yield (%)

tration difference inside and outside plant cells, which

1.5
1 al., 2003). So using high ratio of material/ solvent could
0.5
0
1:05 1:10 1:15
Material/solvents ratio
Figure 2. E ect of material/solvents ratio on
extraction yield of FML.
increased significantly. Further increase of ethanol
concentration 80% was only affected in slightly increase
of extraction yield. In addition, when ethanol concen-
tration increased from 80 - 100 %, however, extraction
yield slightly decreased. It was in accordance with existed
studies (Chen et al., 2008). Besides, the color of extracts
deepened gradually when the volume of ethanol
percentage was higher than 60%, which indicated that
more undesirable compounds, primary chlorophyll, were
also extracted. Therefore, it was decided to use ethanol
60% as extraction sovent in the following experiments.
consequently prompted diffusion rate of solute particles
and made more flavonoids molecules enter solution (Li et
help achieve high extraction yield, however large amount
of solvent resulted flavonoids content in the extract was
unnecessary low. Therefore, material/solvent ratio of 1:15
was sufficient for the experiment.
1:20 1:25
Effect of microwave irradiation power and time on
extraction yield of FML
Microwave irradiation power and time were another main
parameter in the extraction procedure (Zhang et al.,
2005). Extractions at different irradiation powers, such as
280, 420, 560 and 700W for 5 min, were tested. Results
are listed in Figure 3. It was found that 560W is the
optimal irradiation powder. Microwave irradiation time
were investigated during 1, 2, 5, 10 min in 560 W, and the
results is represented in Figure 4. Extraction yield was
rapidly increased in the first 5 minutes of extraction.
Further irradiation was only slightly increased extraction
yield. Hence it was unnecessary to carry experiments
with more than 5 min irradiation; 5 min is the optimal
irradiation time.

Effect of material/solvents ratio on extraction yield of Anti-fatigue activity of FML

FML
Material/solvents ratio was another factor affecting
extraction yield of flavonoids (Tang et al., 2005; Fu et al.,
2008), which was set at 1:5, 1:10, 1:15, 1:20 and 1:25
respectively. As shown in Figure 2, extraction yield quick-
ly increased with the increase of material/solvent ratio
from 1:5 - 1:15, afterward, extraction yield slightly
increased with the increase of material/solvent ratio from
Fatigue is the symptom, which indicated that the health is
about or already subjected to harm (Zhang et al., 2008).
Many reports have indicated that flavonoids can provide
energy immediately during exercise and were helpful on
extensive exercise (Cao et al., 2003; Yu et al., 2008).
However, there is no information in detail concerning the
anti-fatigue effect of FML.
In this study, the body weights of mice increased during
 Li et al. 901

Table 1. Effects of FML on body weight of mice (mean SD, n =

24).

Body weight (g)

Group Initial Intermediate Terminal
CG 19.45 0.24 26.12 0.45 30.04 0.36
LG 20.04 0.35 25.87 0.39 30.54 0.52
MG 20.42 0.17 26.25 0.51 29.77 0.63
HG 19.76 0.22 25.59 0.34 30.16 0.44

Table 2. Effect of FML on blood lactate, serum BUN and hepatic glycogen of mice (mean SD, n = 8).

blood lactate (mmol/L)

Groups Before swimming After swimming Serum BUN (mmol/L) Hepatic glycogen (mg/g)
CG 4.24 0.51 11.23 1.54 9.86 1.13 7.54 2.87
LG 4.53 0.27 10.89 1.14 9.34 0.78 14.27 3.62
MG 4.21 0.65 8.15 1.48 8.41 1.06 18.46 3.18
HG 4.50 0.33 8.86 1.74 8.13 0.98 17.54 4.02
*P < 0.05 as compared with CG.

2.5 14 ?
Swimming endurance time ?
2 12
Extraction yield (% )

10 ?
1.5
8
1
6
(min)

0.5
4
0
1 2 5 10 2
Microwave irradiation time (min)
0
Figure 4. Effect of Microwave irradiation time on
CG LG MG HG
extraction yield of FML.
Group

the experimental period (28 days) is shown in Table 1.
There was no significant difference in the body weights of
mice in the three FML groups compared with CG during
initial, intermediate and terminal stages in the experiment
(p > 0.05).
Swimming endurance time of mice is shown in Figure
5. The average swimming time of mice of the treated
groups were all remarkably longer than that of CG (P <
0.05). These results indicated that FML had significant
effects on the endurance of mice in the experiment.
The levels of blood lactate, serum blood urea nitrogen
(BUN) and hepatic glycogen of mice are shown in Table
2. These results revealed that treated groups reduced the
serum BUN and blood lactate levels in mice compared
with CG, and increased the hepatic glycogen level. The
three biochemical parameters of MG and HG had
significant difference (p < 0.05). Hepatic glycogen level of
LG had significant difference (p < 0.05), but the other two
Figure 5. Effects of FML on swimming endurance time of
mice (mean SD, n = 8).
*P < 0.05 as compared with CG.
biochemical parameters had the adverse results (p >
0.05).
Both high-dose and middle-dose of FML could
obviously prolong the swimming endurance time, and had
active effects on the serum BUN, hepatic glycogen and
blood lactic acid levels in mice. It was considered that
appropriate dose of FML possessed an anti-fatigue
activity in mice.
Conclusions
Conditions of MAE method for extracting flavonoids from
902 Afr. J. Agric. Res.
mulberry leaves were studied and established. It can be
concluded that the optimal extraction parameters are as
followings: ethanol concentration is 60%, material/solvent
ratio is 1:15, microwave irradiation power is 560 W and
irradiation time is 5 min. Meanwhile, our results suggest
that flavonoids from mulberry leaves (FML) had signifi-
cant anti-fatigue effects. However further studies to clarify
the detailed mechanisms involved in the anti-fatigue
properties of FML are necessary.
REFERENCES
Barnabas IJ, Dean JR, Owen SP (1994). Supercritical-fluid extraction of
analytes from environmental samples - a review. Analyst. 119: 2381-
2394.
Buchholz KD, Pawliszyn J (1994). Optimization of solid-phase
microextraction conditions for determination of phenols. Analytical
Chemistry. 66: 160-167.
Cao WG, Liu ZQ, Shao Y, Tao YD (2003). A progress in pharmacological
research of flavonoids. Acta Botanica Boreali-occidentalia Sinica. 23:
2241-2247.
Chen JJ, Li XG (2007). Hypolipidemic effect of flavonoids from mulberry
leaves in triton WR-1339 induced hyperlipidemic mice. Asia Pacific J.
.Clinical. Nutrition. 16: 290-294.
Chen SS, Spiro M (1995). Kinetics of microwave extraction of rosemary
leaves in hexane, ethanol and a hexane + ethanol mixture. Flavour
and Fragrance J. 10: 101-112.
Chen YH, Wang JP, Jiang HQ (2008). Optimization of Extraction
Technology of Total Flavonoids from Mulberry Leaves by Orthogonal
Design. Food and Drug. 3: 17-18.
Dai JM, Yaylayan VA, Raghavan GSV, Pare JR, Dai JM (1999).
Extraction and colorimetric determination of azadirachtin-related
limonoids in neem seed kernel. J. Agric. Food. Chem. 47: 3738-
3742.
Fu WL, Yang LG, Sun GJ (2008). Microwave-assisted extraction of
flavonoids from chrysanthemum. Food. Res. Dev. 29: 1-3.
Hawthorne SB, Miller DJ (1994). Direct comparison of Soxhlet and low-
temperature and high-temperature supercritical CO2 extraction
efficiencies of organics from environmental solids. Analytical Chem.
66: 4005-4012.
Heemken OP, Theobald N, Wenclawiak BW (1997). Comparison of ASE
and SFE with Soxhlet, sonication, and methanolic saponification
extractions for the determination of organic micropollutants in marine
particulate matter. Analytical Chem. 69: 2171-2180.
Jia ZS, Tang MC, Wu JM (1999). The determination of flavonoid
contents in mulverry and their scavenging effects on superoxide
radicals. Food. Chem. 64: 555-559.
Kerem Z, German-Shashoua H, Yarden O (2005). Microwave-assisted
extraction of bioactive saponins from chickpea (Cicer arietinum L). J.
Sci. Food. Agric. 85: 406-412.
Lan DLXP (2007). Study on the Extracting Condition of the Total
Flavonoid from Ipomoea batatas Lam leaves. Studies of trace
elements and health. 1: 47-48.
Lapvetelainen T, Tiihonen A, Koskela P, Nevalainen T, Lindblom J, Kiraly
K, Halonen P, Helminen HJ (1997). Training a large number of
laboratory mice using running wheels and analyzing running behavior
by use of a computer-assisted system, Lab. Anim. Sci. 4: 172-179.
Lee CY, Sim SM, Cheng HM (2007). Systemic absorption of
antioxidants from mulberry (Morus alba L) leaf extracts using an in
situ rat intestinal preparation. Nutr. Res. (N. Y., NY, U. S.) .27: 492-
497.
Li MJ, You JY, Liu ZY, Zhang HQ (2003). Microwave-assisted extraction
of flavonoids from flos sophorae. J. Guang Xi Normal University. 21:
103-104.
Li SF, Wen HB (2006). The Functionality of Hydrolysised Corn Protein
Peptide-Study of Anti Fatigue. Food Sci. 27: 245-247.
Llompart MP, Lorenzo RA, Cela RF, Li K, Belanger JMR, Pare JRJ
(1997). Evaluation of supercritical fluid extraction, microwave-
assisted extraction and sonication in the determination of some
phenolic compounds from various soil matrices. J. Chromatogr. 774:
243-251.
Mandal V, Mohan Y, Hemalatha S (2007). Microwave Assisted
Extraction - An Innovative and Promising Extraction Tool for
Medicinal Plant Research. Pharmacog. Rev. 1: 9-17.
Mattina MJI, Berger WAI , Denson CL (1997). Microwave-assisted
extraction of taxanes from Taxus biomass. J. Agric. Food. Chem. 45:
4691-4692.
Middleton E, Kandaswami C, Theoharides TC (2000). The effects of
plant flavonoids on mammalian cells: implications for inflammation,
heart disease and cancer. Pharmacol. ThRev. 52: 673-751.
Orlans FB (1987). Case studies of ethical dilemmas, Lab. Anim. Sci.
Special Issue, pp. 59-64.
Pan X, Liu H, Jia G, Shu YY (2000). Microwave-assisted extraction of
glycyrrhizic acid from licorice root. Biochem. Eng. J. 5: 173-177.
Qian LN, Chen P, Li XL, Xiao YY (2008). Study on the antifatigue
function of total saponins from Panax japonicus. Chinese J. Hospital
Pharm. 28: 1238-1240.
Quan PT, Hang TV, Ha NH, De NX (2006). Microwave-assisted
extraction of polyphenols from fresh tea shoot. Sci. Technol. Dev. 9:
69-75.
Shi YL, Xin XL, Yang L, Cai DH (2004). Effect of Lentinan on Anti-
Exhaustion and Liver Protection of Exhausted Mice. J. Jilin Agric.
University. 26: 301-304.
Shu YY, Ko MY, Chang YS (2003). Microwave-assisted extraction of
ginsenosides from ginseng root. Microchem. J. 74: 131-139.
Snijman PW, Swanevelder S, Joubert E (2007). The antimutagenic
activity of the major flavonoids of rooibos (Aspalathus linearis): Some
doseresponse effects on mutagen activationflavonoid interactions.
Mutation Res. 631: 111-123.
Tang KW, Yi JM, Zhang YC (2005). Microwave-Assisted Process for
Extraction of Flavonoid from Artemisia selengensis. Nat. Prod.
Res.Dev. 17: 622-624.
Tian M, Yan HY, Row KH (2008). Extraction of Glycyrrhizic Acid and
Glabridin from Licorice. Int. J. Mole. Sci. 9: 571-577.
Tu YS, Bi XL (2005). Application of Microwave - Assisted Extraction
Technology to Study of Extraction of Traditional Chinese Medicine
and Medicine from Natural Substances. World Science Technology-
Modernization of Traditional Chinese Medicine. 3: 65-70.
Wang J, Wu FA, Zhao H, Liu L , Wu QS (2008). Isolation of flavonoids
from mulberry (Morus alba L.) leaves with macroporous resins,
African J. Biotechnol. 7: 2147-2155.
Wang QA, Zhou B, Shan Y. (2004). Progress on antioxidant activation
and extracting technology of flavonoids. Chem. Product. Technol. 11:
29-33.
Xie W, Yang YL, Zheng HY (2004). Experiment Research Into the
Influence of EGb on the Anti-Movement-Fatigue Ability of Small Mice.
J.l Shandong. Physical. Education. Institute. 20: 51-53.
Xu G (2007). Studies on the extracting and antioxidant activities of
flavonoids in sweet potatoes. J. Food. Sci. Biotechnol. 26: 22-27.
Yu FR, Feng ST, Xie MR (2008). Anti-fatigue effect of cynomorium
songaricum flavone on old rats. Chinese J. Rehabilitation. Theory
Pract. 14: 1141-1143.
Zhang F, Chen B, Xiao S, Yao SZ (2005). Optimization and comparison
of different extraction techniques for sanguinarine and chelerythrine
in fruits of Macleaya cordata (Willd) R. Br.. Separation Purification
Technol. 42: 283-290.
Zhang GY, Zhao HJ, Xin ZJ, Qu JL, Wang BG, Wang H (2008). Effect of
maple syrup on fatigue resistance in mice. J. Xi an Jiaotong
University (Medical Sci.). 29: 644-646.